Project 1: Adenosine Deaminase Deficiency Genetic deficiency of adenosine deaminase (ADA) can result in severe T, B and NK cell lymphopenia, but also non-immunological abnormalities of the skeleton, liver, lungs, kidney and central nervous system. We follow a cohort of 40 ADA-deficient patients in whom we have recently noted additional remarkable hematologic features that include neutropenia and dysplastic changes in peripheral blood and bone marrow examinations. These findings are associated to prolonged marrow suppression after low-dose chemotherapy and I.V. antibiotic-induced neutropenia (Sokolic R., Maric I., Kesserwan C., Garabedian E., Hanson C.I., Dodds M., Buckley R., Issekutz A., Kamani N., Shaw K., Tan B., Bali P., Hershfield M.S., Kohn D.B., Wayne A.S., Candotti F.: Myeloid dysplasia and bone marrow hypocellularity in adenosine deaminase-deficient severe combined immune deficiency. Blood 2011; 118:2688-94). We hypothesize that these myelodysplastic features underline a yet unrecognized hematopoietic stem cell defect due to ADA deficiency. To test this hypothesis, we will take advantage of the available Ada KO mice and perform in vitro (CFUs) and in vivo (competitive repopulation assays) analysis of stem/progenitor cell potential. We will also assess whether ADA deficiency results in hematopoietic defects in developing zebrafish. We have also observed a high prevalence of dermatofibrosarcoma protuberans (DFSP) among our ADA-SCID patients (Kesserwan C., Sokolic R., Cowen E.W., Garabedian E.K., Heselmeyer-Haddad K., Lee C.R., Pittaluga S., Ortiz C., Baird K., Lopez-Terrada D., Bridge J.A., Wayne A.S., Candotti F.: Multicentric dermatofibrosarcoma protuberans in patients with adenosine deaminase-deficient severe combined immune deficiency. J Allergy Clin Immunol 2012; 129:762-9). DFSP accounts for 0.1% of all cancers and only about 180 pediatric cases have been reported. The striking association between two rare conditions suggests the possibility of a shared cause. Because adenosine plays an important role in the development of fibrosis and ADA deficiency predisposes to increased susceptibility to DNA strand breaks, we postulate that the combination of the fibrogenic milieu induced by increased adenosine levels and the intrinsic genomic instability of ADA-deficient cells may be responsible for the high risk of DFSP. To test this hypothesis, we will assess the rate of gross chromosomal rearrangement and analyze chromosomal segregation in S. cerevisiae deleted of AAH1, the yeast homolog of the human ADA gene. In addition, we will to assess genomic instability in primary lymphocytes and lymphoblastoid cell lines from ADA-deficient patients by measuring their sensitivity to radiation and methyl methanesulfonate (MMS) treatment. The natural history of multicentric DFSP in ADA-SCID patients is unclear and presents significant clinical management challenges. We will monitor the natural history of these unusual tumors through a combination of conservative surgical approach, close observation and experimental evaluation of topical administration of receptor tyrosine kinase inhibitors. Project 2: Wiskott-Aldrich Syndrome (WAS) WAS is a rare X-linked disorder caused by mutations of the WAS gene and presenting with life-threatening hemorrhages, infections, autoimmunity and cancer. For yet unclear resons, WAS patients shoe high frequency of spontaneous reversion events. We have recently shown that the general mutation rate is not increased in WAS compared to controls (Davis B.R., Yan Q., Bui J.H., Felix K., Moratto D., Muul L.M., Prokopishyn N.L., Blaese R.M., Candotti F.: Somatic Mosaicism in the Wiskott-Aldrich Syndrome: Molecular and Functional Characterization of Genotypic Revertants. Clin Immunol 2010; 135:72-83). Autoimmune disorders are diagnosed in 40-70% of WAS patients and complicate post-HSC transplant outcome in >20% of cases. We and others have characterized the CD4+CD25+FOXP3+ natural regulatory T cells (nTregs) in Was KO mice and WAS patients and shown abnormal capabilities of WAS nTreg to suppress T and B cell proliferation, which may contribute to autoimmunity (Adriani M., Jones K.A., Uchiyama T., Kirby M., Silvin C., Anderson S., Candotti F.: Defective inhibition of B-cell proliferation by Wiskott-Aldrich syndrome protein-deficient regulatory T cells. Blood 2001; 117:6608-11). A level of complexity is added by the existence of a second group of Treg cells that are induced in the periphery (iTreg) in the appropriate cytokine milieu (IL-2+TGF-b). We hypothesize that the iTreg cell function may be affected in WAS. To test this hypothesis, Was KO iTreg cells will be generated and tested in T and B cell suppression assays. In vivo studies will also be conducted to evaluate the capability of Was KO iTreg to prevent the lethal autoimmune syndrome in Scurfy mice. Was KO mice are susceptible to autoimmune disorders, such as inflammatory bowel disease, IgA nephropathy and production of antinuclear antibodies (Shimizu M., Nikolov N.P., Ueno K., Ohta K., Siegel R.M., Yachie A., Candotti F.: Development of IgA nephropathy-like glomerulonephritis associated with Wiskott-Aldrich syndrome protein deficiency. Clin Immunol 2012; 142:160-6). These features will allow testing pharmacological intervention aiming at altering the equilibrium between immune suppressing Treg cells and pro-inflammatory Th17 cells as a potential therapeutic option. In addition to experiments in mice, we will characterize the suppression defect of WAS nTregs on B lymphocyte proliferation and the ability of generating functional iTreg using T cells isolated from WAS research subjects with different clinical presentations in terms of autoimmune complications and presence or absence of auto-antibodies. Project 3: Reticular Dysgenesis (RD) RD represents less than 3% of all SCID and is characterized by profound neutropenia and severe T-cell lymphopenia. Affected newborns often show sensorineural deafness. We have recently shown that RD is caused by mutations of the adenylate kinase-2 (AK2) gene (Lagresle-Peyrou C., Six E.M., Picard C., Rieux-Laucat F., Michel V., Ditadi A., Demerens-de Chappedelaine C., Morillon E., Valensi F., Simon-Stoos K.L., Mullikin J.C., Noroski L.M., Besse C., Wulffraat N.M., Ferster A., Abecasis M.M., Calvo F., Petit C., Candotti F., Abel L., Fischer A., Cavazzana-Calvo M.: Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness. Nat Genet 2009; 41:106-11). AK2 is expressed in the mitochondrial intermembrane space in a variety of tissues such as the liver, kidney, spleen and heart, where it regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP+AMP. The molecular mechanisms responsible for the biological defects of the disease remain unclear. We set out to develop mouse and zebrafis models of the disease. Ak2 conditionally targeted mice will be generated and crossed to Cre deleter strains, such as Lck-Cre, CD19-Cre, Lyz2-Cre, Mx-Cre, CMV-Cre. Resulting animals will be characterized for development and function of the myeloid and lymphoid compartments. We are also generating zebrafish models, including Ak2 knockdown morphants and Ak2 mutant fish. The latter will also be recovered from an ENU-mutagenized zebrafish library that contains an Ak2 missense mutation with predicted severe functional effects and by using zinc finger nuclease-mediated gene targeting. We will also generate induced pluripotent stem (iPS) cells starting from skin biopsies of RD patients. RD iPS will be differentiated into hematopoietic progenitors and mature T, B, NK and myeloid cells. The results from these experiments will characterize the hematological differentiation steps that are affected by AK2 deficiency and provide a framework for preclinical studies of AK2 gene correction.